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Formaldehyde dehydrogenase genes from methylotrophic yeasts

a technology of methylotrophic yeast and dehydrogenase, which is applied in the field of methylotrophic yeast, can solve the problems of inconvenient monitoring of methanol concentration, insufficient promotion or convenience in all settings, and induction of psub>aox1/sub>-controlled expression strains in large-volume high-density fermentor cultures, etc., and achieves the effect of reducing the risk of toxicity and reducing the risk o

Inactive Publication Date: 2007-07-31
RES CORP TECH INC
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  • Claims
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AI Technical Summary

Benefits of technology

[0021]In addition, the present invention provides a kit which comprises an expression vector comprising an FLD gene as a selectable marker gene and an expression cassette. The expression cassette comprises a promoter and a 3′ termination sequence...

Problems solved by technology

Although many proteins have been successfully produced using PAOX1, this promoter is not appropriate or convenient in all settings.
For example, in shake-flask cultures, methanol rapidly evaporates, and it is inconvenient to monitor methanol concentrations and repeatedly add the compound to the medium.
In addition, the storage of large amounts of methanol needed for the growth and induction of PAOX1-controlled expression strains in large-volume high-density fermentor cultures is a potential fire hazard.

Method used

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  • Formaldehyde dehydrogenase genes from methylotrophic yeasts
  • Formaldehyde dehydrogenase genes from methylotrophic yeasts
  • Formaldehyde dehydrogenase genes from methylotrophic yeasts

Examples

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example 1

Isolation of Formaldehyde Dehydrogenase-defective Mutants of P. pastoris

[0095]As a first step in cloning the P. pastoris formaldehyde dehydrogenase gene (FLD1), mutants were sought that were specifically defective in FLD activity. Previous biochemical studies of methylotrophic yeasts indicated that FLD is involved in the metabolism of both methanol as carbon source and methylamine as nitrogen source (Zwart et al., 1983). To search for P. pastoris fld1 mutants, nitrosoguanidine-mutagenized cultures were screened for strains that were unable to utilize methanol as carbon source and methylamine as nitrogen source. Complementation analysis and other classical genetic techniques were performed as described in Cregg and Russell (1998). Five mutants belonging to a single complementation group were identified.

[0096]These five strains were further examined by measuring the levels of activity of key methanol pathway enzymes in extracts prepared from methanol-induced cultures of each strain. ...

example 2

Isolation and Characterization of the P. pastoris FLD1 Gene

[0099]To clone the putative FLD1 gene by functional complementation, strain GS241 was first crossed to P. pastoris strain GS115 (his4) to obtain a derivative that was both methanol-utilization defective (Mut−) and auxotrophic for histidine (His−). One Mut−His− strain that resulted from this cross, MS105 (fld1-1 his4), was then transformed with 5–10 μg of a P. pastoris genomic DNA library constructed in the P. pastoris-E. coli shuttle vector pYM8 using the spheroplast method (Cregg et al., 1985; Liu et al., 1995). The plasmid pYM8 is composed of the Saccharomyces cerevisiae histidinol dehydrogenase gene (SHIS4) and a P. pastoris-specific autonomous replication sequence (PARS1) inserted into E. coli plasmid pBR322. Approximately 50,000 library transformants were selected for His+ prototrophy on YND medium agar and resultant selected clones further selected on YNM plates for Mut+ phenotype. Total DNA was extracted from a pool o...

example 3

Isolation and Characterization of the Hansenula polymorpha FLD Gene

[0105]The putative H. polymorpha FLD1 gene was isolated using the same functional complementation strategy described above for the P. pastoris gene. An H. polymorpha genomic DNA library was constructed in P. pastoris vector pYM8 in the same manner as the P. pastoris library (Liu et al. 1995). Briefly, H. polymorpha genomic DNA was partially digested with Sau3A and size selected for fragments of 5–20 kb. These fragments were ligated into the BamHI site of pYM8. The library was composed of approximately 100,000 independent E. coli transformants with greater than 90% containing an insert. The average size of insert DNA was approximately 10 kb. Assuming that the size of the H. polymorpha genome is 10,000 kb, the library contained approximately 100 genome equivalents of H. polymorpha genomic DNA. Plasmids were recovered and analyzed for ones that were capable of simultaneously retransforming MS105 (fld1-1 his4 to both His...

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Abstract

The present invention provides formaldehyde dehydrogenase genes (FLD) from methylotrophic yeasts. The FLD structural genes confer resistance to formaldehyde and are therefore useful as a selectable marker in methylotrophic yeasts. The FLD promoter sequences are strongly and independently induced by either methanol as sole carbon source (with ammonium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source). Induction under either methanol, methylamine or both provides levels of heterologous gene expression comparable to those obtained with the commonly used alcohol oxidase I gene promoter (PAOX1). The FLD promoter of Pichia pastoris (PFLD1) is an attractive alternative to PAOX1 for expression of foreign genes in P. pastoris, allowing regulation by carbon (methanol) or nitrogen (methylamine) source within the same expression strain. Yeast strains, expression cassettes, expression vectors, and host cells comprising an FLD gene promoter and 3′ termination sequence are also provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present application is a divisional of Ser. No. 09 / 345,828, filed Jul. 2, 1999, now U.S. Pat. No. 6,730,499, issued May 4, 2004, which claims the benefit of Provisional Application Ser. No. 60 / 091,699, filed Jul. 3, 1998.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This work was supported in part from grants from the U.S. National Institutes of Health (DK43698) and the U.S. National Science Foundation (MCB-9514289). The government may have rights in the invention.BACKGROUND OF THE INVENTION[0003]Pichia is a methylotrophic yeast that is widely used for the production of heterologous proteins of industrial and academic interest (Cregg, 1998; Higgins and Cregg, 1998). FLD is an important enzyme in the utilization of methanol as a carbon and energy source (Veenhuis et al., 1983). In methylotrophic yeasts, the methanol metabolic pathway is thought to be nearly the same, beginning with the oxidation of methanol to fo...

Claims

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Application Information

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IPC IPC(8): C07H21/04C12N1/00C12N1/16C12N15/00C12N9/02C12N15/81
CPCC12N9/0008C12N15/815
Inventor CREGG, JAMES M.
Owner RES CORP TECH INC
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